Remarkable advances have been made in hemidesmosomal biology and biochemistry since the initial report by our group that this organelle is the target of autoantibodies produced by patients with Bullous Pemphigoid (BP) and Herpes Gestationis (HG). In studies supported by this R29 award, patient autoantibodies were used as specific probes to facilitate the molecular cloning and characterization of antigens associated with these autoimmune diseases. One antigen, BP18O, was of particular interest because of its unique set of structural features. The BP180 antigen was found to be a transmembrane protein with an N-terminal segment embedded in the cytoplasmic plaque of the epidermal hemidesmosome and a long C-terminal collagenous ectodomain that projects into the basal lamina. Based on primary structural information the BP180 ectodomain is predicted to form a homo- or heterotypic collagen-like triple helix that may stably interact with one or more components of the extracellular matrix. The N-terminal cytoplasmic domain of BP180 is postulated to link up with the intermediate filament network, either through direct association with the keratins or via a system of linker molecules. The present proposal represents a logical extension of these previous investigations - an in-depth functional analysis of this novel protein. The proposed studies include a broad range of experimental strategies, combining tools from the areas of molecular genetics, immunology, biochemistry and cell biology. Much of the effort will be directed toward the manipulation and analysis of living systems - cultured cells, organ explants and whole animals. BP18O "loss-of-function" strategies include the use of specific antibodies and peptides as functional inhibitors, and various antisense techniques to inhibit BP180 expression. In addition, mutated forms of BP18O will be expressed in cultured cells (transfectants) and whole animals (transgenics) in an attempt to create a dominant negative phenotype. BP180 "gain-of-function" will be accomplished by introducing "wild type" BP180, via gene transfer technology, into cells that do not normally express this protein. The genetically and biochemically manipulated cells will be assayed for alterations in, or emergence of, cellular phenomena related to cell- matrix or cell-cell adhesion, proliferation, differentiation, or the organization of hemidesmosomal or cytoskeletal structures. Information obtained through the manipulation of BP18O in situ will be confirmed and extended using well-established ex vivo biochemical assays. The long- range goal of this proposed project is to elucidate, at the molecular level, the structural and functional properties of this novel hemidesmosomal protein. The information derived from these studies will further our understanding of epithelial-stromal interactions under normal and pathological states.